Project Summary/Abstract Leucine rich repeat kinase 1 (Lrrk1) belongs to ROCO family proteins and contains three ankyrin repeat, seven leucine-rich repeat, a GTPase-like domain of Roc (Ras of complex proteins), COR (C-terminal of Roc) domain, and a serine/threonine kinase domain that is regulated by GTP binding to the Roc domain. We recently demonstrated that Lrrk1 knockout (KO) mice as well as a patient with a Lrrk1 mutation are severely osteopetrotic resulting from mature OC dysfunction and reduced bone resorption in the metaphyseal trabecular regions of the tubular and axial bones. These observations make Lrrk1 an ideal drug target for treatment of high bone turnover. Our focus in this grant is to develop small molecular weight inhibitors of Lrrk1 for treatment and prevention of osteoporosis and osteoporotic fractures. To this end, we propose two hypotheses in this study: 1) Lrrk1 inhibitor, IN04, inhibits elevated bone resorption (BR) but not bone formation (BF) in OVX mice; 2) INO4 inhibits Lrrk1 kinase activity by directly binding to the active site of the Lrrk1 kinase domain. To test the hypothesis 1, experiments are designed to compare the systemic effects of IN04 versus bisphosphonates on bone mass and strength in OVX- and Sham mice, by microCT, histomorphometric analyses, and mechanical property tests, and measure BR, BF markers in serum from vehicle treated or IN04 treated OVX mice at various time points. OC-secreted coupling factors in IN04-, bisphosphonates- or vehicle-treated OCs will also be examined by real-time PCR and Western blot analyses. To test the hypothesis 2, experiments are designed to express and purify recombinant human Lrrk1 kinase domain produced in E. coli cells, co-crystalize this kinase domain with ATP and IN04, and resolve the crystal structures. More potential Lrrk1 inhibitors will be screened by re-docking small molecules into the active pocket of the resolved Lrrk1 kinase domain. The results of this application will develop potential small molecular weight inhibitors of Lrrk1 for treatment and prevention of osteoporosis.